IsoPlexis proposes to deliver a novel multi-omic method for targeted profiling of both the TCR sequence and proteome from an array of 1000+ single cells. Specifically, we will deliver a single-cell, TCR sequencing and protein capture assay for identifying responsive antigen specific TCRs, and concurrently evaluate these T-cells for functional response to that antigen. The challenge remains to link the activation of quiescent T-cell embedded in tumors by combination immunotherapies to patient outcome. Determining the combination of therapies to which each individual patient best responds indicates the best course of treatment. The quality of single-cell polyfunctional response of these immune cells correlates to positive outcomes far better than traditional bulk analysis. For example, PD-1 is upregulated upon T-cell activation while PD-L1 is expressed by a range of cell types. Since PD-1/PD-L1 interactions negatively regulate T cell immune function, PD-1/PD-L1 blockade can rescue effector T cell function. Critical to analyzing TILs is to assess (1) these T-cells? function in the tumor environment in order to enable trial leaders to predict responders vs non-responders, a critical problem in immuno-oncology, and (2) to understand the TCR Sequence of the highest functioning cells. IsoPlexis single- cell secretion analysis exceeds its competition in the generation and quantitation of highly-multiplexed, single- cell data. Additional single-cell data from the TCR sequence would help to link antigen specificity to polyfunctional T cells involved in patient response, for improved biomarkers and targeted T-cell therapy development. We propose the following specific aims: (1) develop SCBC flow cell for the dual capture of multiplexed proteins and transcriptome on-device. (2a) produce a miniaturized and benchtop automated instrument of the existing instrument for multi-omic applications. 2b) develop a software suite for automated data processing and intuitive integrated informatics of polyfunctional and transcriptome data. 3) Establish patient learning of phenotype & genotype information in multiple trials, applied with machine learning of large patient genotype/phenotype data. At the end of our Phase II grant, we will demonstrate a dual TCR/proteomic assay on a fully-automated miniaturized SCBC instrument, the IsoMini, and software suite that will be successfully used across three combination therapy trials at Yale, Stanford and Fred Hutch.